The polypeptide hirudin, which was originally isolated from the leech Hirudo medicinalis, is a highly specific thrombin inhibitor possessing a broad therapeutic potential (F. Markwardt, Biomed. Biochim. Acta 44 (1985) 1007-1013). However, it is only possible to prepare the quantities which are required by the recombinant route using transformed microorganisms. In this context, it has been found that the yeast Saccharomyces cerevisiae is suitable as a host organism for producing hirudin which is correctly folded and fully active (EP A1 168 342, EP A1 200 655).
The gene for the alcohol dehydrogenase isoenzyme II (ADH II) is strictly regulated in yeast cells. The product of the ADH II gene is not found when fermentable carbon sources, such as glucose, are added to the fermentation medium. ADH II catalyzes the dehydration of ethanol (C.sub.2 H.sub.5 OH) to acetaldehyde (CH.sub.3 COH). However, conditions for inducing the ADH II promoter can be achieved in simple aerobic batch processes, in shaking flasks or in fermenters, which are started, for example, using a glucose concentration of 4%. When growth takes place on glucose, the so-called "Crabtree" effect results in ethanol being formed initially with the aid of the enzyme ADH I, with the ethanol in turn being used as an additional carbon source once the glucose has been completely consumed. After the glucose has been broken down, the ethanol-degrading enzyme ADH II is induced and the expression of the ADH II gene product begins. If the expression of a gene encoding a heterologous protein is under control of the ADH II promoter, expression of the heterologous protein will begin once the glucose has been metabolized.
As a rule, such batch fermentations do not provide the high yield which is sought for industrial applications. In general, industrial applications require a high yield in a short period of time. What is wanted is the separation of the fermentation into a growth phase and a production phase, as is achieved in classical antibiotic fermentations, for example, by means of a limited fed-batch procedure (also known in the art as feed-batch), in which, after a certain cell density has been established, a carbon source is then fed in a growth-limiting manner in order to form the product (e.g. the secondary metabolite penicillin) at high yield under optimal physiological conditions See Hersbach et al.: The Penicillins: Properties, Biosynthesis and Fermentation, in: Biotechnology of Industrial Antibiotics, pp. 45-140, Ed. E. J. Vandamme, Marcel Dekker, New York, 1984.
Because of the way it is regulated, the ADH II promoter system has been used for the preparation of recombinant proteins in the bakers yeast Saccharomyces cerevisiae. See Price et al.: Methods of Enzymology, Vol. 185, 308-318, 1990, which is hereby incorporated by reference. The fermentation system used by Price depends on the growth of the yeast cells and on the natural elimination of glucose as a carbon source. In the absence of glucose, product formation is switched on and continues until the end of the fermentation. However, the yield of product appears to be less than was hoped.
T.o slashed.ttrup et al. (Biotechnol. Bioeng., 35, 339-348, 1990) describe a modified fermentation procedure for the intracellular production of a human insulin fusion protein, which procedure involves feeding with glucose followed by feeding with ethanol in order to ensure optimal induction of the system. This group used a hybrid promoter, which consisted of the ADH II promoter ligated to the glyceraldehyde-3-phosphate dehydrogenase promoter. These workers succeeded in almost doubling the volume-related product yield (grams protein/liter), as compared with a batch process, by means of continually feeding glucose at a constant rate over the entire fermentation period. A further doubling was finally achieved by means of replacing the glucose feed, from a particular time point onward, with an ethanol feed. It was concluded from this result that even when there is no glucose, or very low glucose concentrations of less than 20 mg/l, in the culture broth, the hybrid promoter is still partially repressed by glucose or a metabolite of glycolysis. Complete induction was only achieved by adding ethanol.
Thus, it would be useful to have a high-yield process for producing heterologous protein in yeast using the ADH II promoter system. As used herein an "ADH II promoter system" means that the expression of a heterologous gene is under the control of the ADH II promoter.